Torque teno virus (TTV) was first discovered in a Japanese patient with post-transfusion non-A-E hepatitis in 1997 (Nishizawa, T., Okamoto, H., Konishi, K., Yoshizawa, H. Miyakawa, Y., and Mayumi, M. (1997). A novel DNA virus (TTV) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology. Biochem Biophys Res Commun 241(1), 92-7.). Since then, a large number of human TTV strains and two groups of TTV-related viruses, designated subsequently as Torque teno mini virus (TTMV) and Torque teno midi virus (TTMDV), have been identified with high prevalence in serum and other tissues from healthy humans (Hino, S., and Miyata, H. (2007). Torque teno virus (TTV): current status. Rev Med Virol 17(1), 45-57; Okamoto, H. (2009a). History of discoveries and pathogenicity of TT viruses. Curr Top Microbiol Immunol 331, 1-20). Human TTV, TTMV and TTMDV are non-enveloped spherical viruses with circular single-stranded DNA (ssDNA) genomes of 3.6-3.9, 2.8-2.9 and 3.2 kb in length, respectively, and they are currently classified into a newly-established family Anelloviridae by the International Committee on Taxonomy of Viruses (ICTV (Biagini, P. (2009). Classification of TTV and related viruses (anelloviruses). Curr Top Microbiol Immunol 331, 21-33). These three groups of TTV-related viruses exhibit a high degree of genetic heterogeneity, each consisting of many genogroups and genotypes (Biagini, P., Gallian, P., Cantaloube, J. F., Attoui, H., de Micco, P., and de Lamballerie, X. (2006). Distribution and genetic analysis of TTV and TTMV major phylogenetic groups in French blood donors. J Med Virol 78(2), 298-304; Jelcic, I., Hotz-Wagenblatt, A. , Hunziker, A., Zur Hausen, H., and de Villiers, E. M. (2004). Isolation of multiple TT virus genotypes from spleen biopsy tissue from a Hodgkin's disease patient: genome reorganization and diversity in the hypervariable region. J Virol 78(14), 7498-507). The prevalence of multiple infections of TTV with different genotypes as well as dual or triple infections of TTV, TTMV and TTMDV have been documented in humans, and are considered to be a common event in healthy human adults (Niel, C., Saback, F. L., and Lampe, E. (2000). Coinfection with multiple TT virus strains belonging to different genotypes is a common event in healthy Brazilian adults. J Clin Microbiol 38(5), 1926-30; Ninomiya, M., Takahashi, M., Hoshino, Y., Ichiyama, K., Simmonds, P., and Okamoto, H. (2009). Analysis of the entire genomes of torque teno midi virus variants in chimpanzees: infrequent cross-species infection between humans and chimpanzees. J Gen Virol 90(Pt 2), 347-58; Okamoto, H. (2009a). History of discoveries and pathogenicity of TT viruses. Curr Top Microbiol Immunol 331, 1-20; Takayama, S., Miura, T., Matsuo, S., Taki, M., and Sugii, S. (1999). Prevalence and persistence of a novel DNA TT virus (TTV) infection in Japanese haemophiliacs. Br J Haematol 104(3), 626-9).
TTV infects not only humans but also various other animal species as well including non-human primates, tupaias, pigs, cattle, cats, dogs and sea lions (Biagini, P., Uch, R., Belhouchet, M., Attoui, H., Cantaloube, J. F., Brisbane, N., and de Micco, P. (2007). Circular genomes related to anelloviruses identified in human and animal samples by using a combined rolling-circle amplification/sequence-independent single primer amplification approach. J Gen Virol 88(Pt 10), 2696-701; Inami, T., Obara, T., Moriyama, M., Arakawa, Y., and Abe, K. (2000). Full-length nucleotide sequence of a simian TT virus isolate obtained from a chimpanzee: evidence for a new TT virus-like species. Virology 277(2), 330-5; Ng, T. F., Suedmeyer, W. K., Wheeler, E., Gulland, F., and Breitbart, M. (2009). Novel anellovirus discovered from a mortality event of captive California sea lions. J Gen Virol 90(Pt 5), 1256-61; Okamoto, H. (2009b). TT viruses in animals. Curr Top Microbiol Immunol 331, 35-52; Okamoto, H., Nishizawa, T., Takahashi, M., Tawara, A., Peng, Y., Kishimoto, J., and Wang, Y. (2001). Genomic and evolutionary characterization of TT virus (TTV) in tupaias and comparison with species-specific TTVs in humans and non-human primates. J Gen Virol 82(Pt 9), 2041-50; Okamoto, H., Nishizawa, T., Tawara, A., Peng; Y., Takahashi, M., Kishimoto, J., Tanaka, T., Miyakawa, Y., and Mayumi, M. (2000a). Species-specific TT viruses in humans and nonhuman primates and their phylogenetic relatedness. Virology 277(2), 368-78; Okamoto, H., Takahashi, M., Nishizawa, T., Tawara, A., Fukai, K., Muramatsu, U., Naito, Y., and Yoshikawa, A. (2002). Genomic characterization of TT viruses (TTVs) in pigs, cats and dogs and their relatedness with species-specific TTVs in primates' and tupaias. J Gen Virol 83(Pt 6), 1291-7). In addition, chimpanzees are also infected with TTMV and TTMDV (Ninomiya, M., Takahashi, M., Hoshino, Y., Ichiyama, K., Simmonds, P., and Okamoto, H. (2009). Analysis of the entire genomes of torque teno midi virus variants in chimpanzees: infrequent cross-species infection between humans and chimpanzees. J Gen Virol 90(Pt 2), 347-58; Okamoto et al., 2000a, supra). Although the genomic sizes of the identified animal TTV strains, especially non-primate animal TTV, are relatively smaller than that of human TTV, they share the same genomic structure with a minimum of two partially overlapping open reading frames (ORF1 and ORF2) translated from the negative ssDNA as well as a short stretch of untranslated region (UTR) with high GC content (˜90%) (Okamoto, 2009b, supra). The arrangement of TTV ORFs is quite similar to that of chicken anemia virus (CAV) belonging to the genus Gyrovirus in the family Circoviridae but is different from porcine circovirus (PCV) types 1 (PCV1) and 2 (PCV2), which are also classified into the same family (Davidson, I., and Shulman, L. M. (2008). Unraveling the puzzle of human anellovirus infections by comparison with avian infections with the chicken anemia virus. Virus Res 137(1), 1-15; Hino, S., and Prasetyo, A. A. (2009). Relationship of Torque teno virus to chicken anemia virus. Curr Top Microbiol Immunol 331, 117-30). The genomes of PCV1 and PCV2 are ambisense, in which the ORF1 is coded for by the genomic strand and the ORF2 is coded for by the antigenomic strand (Hino and Miyata, 2007, supra). Recently, the transcription pattern and translated products of both human TTV genotypes 1 and 6 have been identified by transfection of the respective TTV infectious DNA clones into cultured cells (Mueller, B., Maerz, A., Doberstein, K., Finsterbusch, T., and Mankertz, A. (2008). Gene expression of the human Torque Teno Virus isolate P/1C1. Virology 381(1), 36-45; Qiu, J., Kakkola, L., Cheng, F., Ye, C., Soderlund-Venermo, M., Hedman, K., and Pintel, D. J. (2005). Human circovirus TT virus genotype 6 expresses six proteins following transfection of a full-length clone. J Virol 79(10), 6505-10). Expression of at least six proteins, designated ORF1, ORF2, ORF1/I, ORF2/2, ORF1/2 and ORF2/3, from three or more spliced mRNAs, have been reported (Kakkola, L., Hedman, K., Qiu, J., Pintel, D., and Soderlund-Venermo, M. (2009). Replication of and protein synthesis by TT viruses. Curr Top Microbiol Immunol 331, 53-64; Mueller et al., 2008, supra; Qiu et al., 2005, supra). Accordingly, it is likely that, when more data regarding the animal TTV become available, the presumed genome structure of animal TTV will need to be modified.
Although TTV was first identified in a cryptogenic hepatitis patient, subsequent studies were not able to produce evidence of a significant role of TTV in the pathogenesis of hepatitis or other diseases (Hino and Miyata, 2007, supra; Maggi, F., and Bendinelli, M. (2009). Immunobiology of the Torque teno viruses and other anelloviruses. Curr Top Microbiol Immunol 331, 65-90; Okamoto, 2009a, supra). While human TTV is not considered to be directly associated with a disease, porcine TTV (PTTV) was recently shown to partially contribute to the experimental induction of porcine dermatitis and nephropathy syndrome (PDNS) combined with porcine reproductive and respiratory syndrome virus (PRRSV) infection (Krakowka, S., Hartunian, C., Hamberg, A., Shoup, D., Rings, M., Zhang, Y., Allan, G., and Ellis, J. A. (2008). Evaluation of induction of porcine dermatitis and nephropathy syndrome in gnotobiotic pigs with negative results for porcine circovirus type 2. Am J Vet Res 69(12), 1615-22), and also to the experimental induction of postweaning multisystemic wasting syndrome (PMWS) combined with PCV2 infection in a gnotobiotic pig model (Ellis, J. A., Allan, G., and Krakowka, S. (2008). Effect of coinfection with genogroup 1 porcine torque teno virus on porcine circovirus type 2-associated postweaning multisystemic wasting syndrome in gnotobiotic pigs. Am J Vet Res 69(12), 1608-14). The data suggested that porcine TTV is pathogenic in pigs. However, further in-depth studies with a biologically pure form of PTTV virus to definitively characterize the diseases and lesions associated with PTTV infection are needed.
Compared to human TTV, the genomic information of PTTV is very limited. Currently, only one full-length and two near full-length genomic sequences of PTTV are reported from pigs in Japan and Brazil, respectively (Niel, C., Diniz-Mendes, L., and Devalle, S. (2005). Rolling-circle amplification of Torque teno virus (TTV) complete genomes from human and swine sera and identification of a novel swine TTV genogroup. J Gen Virol 86(Pt 5), 1343-7; Okamoto et al., 2002, supra). Among the three known PTTV strains, the Sd-TTV31 and TTV-1p stains were clustered together into the genogroup 1 (PTTV1), whereas TTV-2p was the sole strain classified into the genogroup 2 (PTTV2) (Niel et al., 2005, supra). However, genogroup classification is a vague concept in the taxonomy of virology, and further and more accurate classification of PTTV is needed but can only be performed when more full-length genomic sequences of new PTTV strains representing multiple genotypes become available.
It was previously showed that PTTV infections were widespread in pigs from six different countries including the United States, Canada, Spain, China, Korea and Thailand (McKeown, N. E., Fenaux, M., Halbur, P. G., and Meng, X. J. (2004). Molecular characterization of porcine TT virus, an orphan virus, in pigs from six different countries. Vet Microbiol 104(1-2), 113-7).
Whether porcine TTVs play a significant role in pathogenesis of specific swine diseases is still debatable. In a gnotobiotic pig model, it was shown that PTTV1 infection alone did not develop any clinical diseases but induced mild histological lesions (Krakowka, S, and Ellis, J. A., 2008. Evaluation of the effects of porcine genogroup 1 torque teno virus in gnotobiotic swine. Am J Vet Res 69, 1623-9). Gnotobiotic pigs that were experimentally inoculated with both PTTV1 and porcine reproductive and respiratory syndrome virus (PRRSV) developed clinical porcine dermatitis and nephropathy syndrome (PDNS) (Krakowka, S., Hartunian, C., Hamberg, A., Shoup, D., Rings, M., Zhang, Y., Allan, G. and Ellis, J. A., 2008. Evaluation of induction of porcine dermatitis and nephropathy syndrome in gnotobiotic pigs with negative results for porcine circovirus type 2. Am J Vet Res 69, 1615-22), whereas pigs inoculated with both PTTV1 and porcine circovirus type 2 (PCV2) developed acute postweaning multisystemic wasting syndrome (PMWS) (Ellis et al., 2008, supra). Although PCV2 is considered as the primary causative agent for clinical PMWS or PCV-associated diseases (PCVAD), a higher prevalence of PTTV2 infection in PMWS-affected pigs with low or no PCV2 than that in non-PMWS-affected pigs was observed in Spain (Kekarainen et al., 2006, supra). The data collectively suggest that porcine TTVs may serve as co-factors involved in triggering or exacerbating diseases in pigs.
Porcine TTV has been detected in porcine serum, fecal, saliva, semen and tissue samples of infected pigs, indicating its diverse transmission routes including both horizontal and vertical transmissions (Kekarainen et al., 2007, supra; Pozzuto, T., Mueller, B., Meehan, B., Ringler, S. S., McIntosh, K. A., Ellis, J. A., Mankertz, A. and Krakowka, S., 2009. In utero transmission of porcine torque teno viruses. Vet Microbiol 137, 375-9; Sibila, M., Martinez-Guino, L., Huerta, E., Llorens, A., Mora, M., Grau-Roma, L., Kekarainen, T. and Segales, J., 2009. Swine torque teno virus (TTV) infection and excretion dynamics in conventional pig farms. Vet Microbiol 139, 213-8). However, current detection of porcine TTV infection was mainly based upon conventional PCR assays. Thus far, neither serological assay nor viral culture system has been established. In particular, nested PCR amplifications of the conserved regions in the UTR, of PTTV1 and PTTV2, respectively, developed by a Spanish group, have become widely used (Kekarainen et al., 2006, supra). Since the amount of virus is likely associated' with the severity of clinical diseases, as demonstrated for PCV2-induced PCVAD (Opriessnig, T., Meng, X. J. and Halbur, P. G., 2007. Porcine circovirus type 2 associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis, and intervention strategies. J Vet Diagn Invest 19, 591-615), it will be important to determine the viral load of porcine TTV by quantitative real-time PCR than the presence of TTV DNA by conventional PCR. In addition, real-time PCR is more reliable, rapid and less expensive than conventional PCR. Recently, two TaqMan probe-based real-time PCR assays were described for detection and quantification of two porcine TTV species (Brassard, J., Gagne, M. J., Houde, A., Poitras, E. and Ward, P., 2009. Development of a real-time TaqMan PCR assay for the detection of porcine and bovine Torque teno virus. J Appl Microbiol. Nov. 14, 2009, Epub ahead of print; Gallei, A., Pesch, S., Esking, W. S., Keller, C. and Ohlinger, V. F., 2009. Porcine Torque teno virus: Determination of viral genomic loads by genogroup-specific multiplex rt-PCR, detection of frequent multiple infections with genogroups 1 or 2, and establishment of viral full-length sequences. Vet Microbiol. Dec. 21, 2009, Epub ahead of print). A main drawback of probe-based assays is that the false-negative results may be obtained if the probe-binding sequences contain mutations (Anderson, T. P., Werno, A. M., Beynon, K. A. and Murdoch, D. R., 2003. Failure to genotype herpes simplex virus by real-time PCR assay and melting curve analysis due to sequence variation within probe binding sites. J Clin Microbiol 41, 2135-7). Considering the high degree of heterogeneity among the sequences of known porcine TTV strains, variations in the probe-binding sequences are expected for field strains of PTTVs. The SYBR green-based real-time PCR is an alternative method avoiding this potential problem, in spite of its relatively lower specificity, which provides a universal way to detect and quantify the potential porcine TTV variants. Moreover, melting curve analysis (MCA) following SYBR green real-time PCR ensures reaction specificity and also allows multiplex detection of distinct types of virus (Ririe, K. M., Rasmussen, R. P. and Wittwer, C. T., 1997. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 245, 154-60). MCA-based SYBR green real-time PCR methods have been successfully applied to various human and veterinary viruses (Gibellini, D., Gardini, F., Vitone, F., Schiavone, P., Furlini, G. and Re, M. C., 2006. Simultaneous detection of HCV and HIV-1 by SYBR Green real time multiplex RT-PCR technique in plasma samples. Mol Cell Probes 20, 223-9; Martinez, E., Rieira, P., Sitja, M., Fang, Y., Oliveira, S, and Maldonado, J., 2008. Simultaneous detection and genotyping of porcine reproductive and respiratory syndrome virus (PRRSV) by real-time RT-PCR and amplicon melting curve analysis using SYBR Green. Res Vet Sci 85, 184-93; Mouillesseaux, K. P., Klimpel, K. R. and Dhar, A. K., 2003. Improvement in the specificity and sensitivity of detection for the Taura syndrome virus and yellow head virus of penaeid shrimp by increasing the amplicon size in SYBR Green real-time RT-PCR. J Virol Methods 111, 121-7; Wilhelm, S., Zimmermann, P., Selbitz, H. J. and Truyen, U., 2006. Real-time PCR protocol for the detection of porcine parvovirus in field samples. J Virol Methods 134, 257-60).
Currently, little is known about PTTV-specific humoral response. Since PCR-based assays do not reflect the course of PTTV infection in pigs, an efficient enzyme-linked immunosorbent assay (ELISA) for detection of PTTV serum antibody is necessary to evaluate seroprevalence of PTTV and help characterize the role of PTTV in porcine diseases.
Thus far, no subunit, killed and live vaccines for porcine TTVs are available. It will be desirable and advantageous to express recombinant PTTV capsid proteins from different genotypes for development of PTTV subunit vaccines, and to construct infectious PTTV molecular DNA clones from different genotypes for propagating biological pure form of PTTVs in cell culture system that are used for killed and live vaccines development.